1. Field of the Invention
The present invention relates to a novel antibiotic peptide originated from the ribosomal protein L1 of Helicobacter pylori and a use of the same.
2. Description of the Related Art
Bacterial infection is the most frequent and sometimes a lethal disease in human. Unfortunately, antibiotic resistance of bacteria rises as another problem due to the overuse of antibiotics. In fact, the time for bacteria to show resistance against a new antibiotic is way shorter than the time that takes to develop a novel antibiotic. For example, the life threatening bacteria, Enterococcus faecalis, Mycobacterium tuberculosis, and Pseudomonas aeruginosa, have grown their resistance against almost every antibiotics known so far (Stuart B. Levy, Scientific American, 46-53, 1998).
Antibiotic tolerance is different from antibiotic resistance, which was first notified in Pneumococcus sp. in 1970s and provided an important clue for the mechanism of penicillin (Tomasz et al., Nature, 227, 138-140, 1970). Those bacteria who show tolerance stop growing in the presence of an antibiotic at a moderate concentration but do not die. Tolerance is generated when the activity of an autolytic enzyme, such as autolysin, is inhibited according to the suppression of a cell wall synthesizing enzyme by an antibiotic. Penicillin kills bacteria by activating endogenous hydrolytic enzymes. However, the bacteria reversely inhibits the enzyme activity to survive from the antibiotic treatment.
Such phenomenon of bacteria being antibiotic tolerant is clinically very important because antibiotics are not useful or effective anymore in clinically treating infection when it is impossible to kill such antibiotic tolerant bacteria (Handwerger and Tomasz, Rev. Infec. Dis., 7, 368-386, 1985). In addition, tolerance is considered as the condition precedent to resistance of bacteria, suggesting that even after antibiotic treatment there are strains that can survive. These strains acquire a new genetic element showing antibiotic resistance so that they can survive and continuously grow in the presence of antibiotics. Actually those bacteria that show resistance against antibiotics were confirmed to have tolerance against them as well (Liu and Tomasz, J. Infect. Dis., 152, 365-372, 1985). Therefore, it is necessary to develop a novel antibiotic that can kill the bacteria showing antibiotic resistance.
Tolerance is mainly divided into two groups according to the aspect of mechanism. First is the phenotypic tolerance that occurs when bacteria become slow to grow and is observed in all the bacteria (Tuomanen E., Revs. Infect. Dis., 3, S279-S291, 1986), and second is the genetic tolerance that is caused by mutation and observed in some specific bacteria. Down regulation of autolysin activity is commonly observed in both cases. This regulation is temporary by a foreign stimulus in the phenotypic tolerance, but is permanent in the genetic tolerance since this group has the mutation caused by the alteration of a pathway that regulates cell hemolysis. The simplest genetic tolerance is the one caused by the deficiency of autolysin. However, it is rare to identify such strains that show tolerance by the deficiency of autolysin for unknown reasons. Rather, the clinically observed tolerance is mainly generated in the course of phenotypic regulation of the activity of autolysin (Tuomanen et al., J. infect. Dis., 158, 36-43, 1988).
As explained hereinbefore, it is necessary to develop a novel antibiotic that works independently from autolysin in order to eliminate bacteria that show resistance against the conventional antibiotics.
Bacteria can kill other bacteria by synthesizing a peptide called bacteriocin or small organic molecules. Bacteriocin can be divided into three groups according to the structural characteristics; lantibiotics, nonlantibiotics, and the ones secreted by signal peptides (Cintas et al., J. Bad., 180, 1988-1994, 1998). Animals including insects can produce the peptide antibiotics (Bevins et al., Ann. Rev. Biochem., 59, 395-414, 1990), which are also divided into three groups according to the structure; cysteine-rich β-sheet peptides, α-helical amphiphilic peptides, and proline-rich peptides (Mayasaki et al., Int. J. Antimicrob. Agents, 9, 269-280, 1998). These antibacterial peptides play an important role in host defense and innate immune system (Boman, H. G., Cell, 65:205, 1991; Boman, H. G., Annu. Rev. Microbiol., 13:61, 1995). The structure of these antibacterial peptides varies according to the amino acid sequence. The most common structure is the cysteine free amphiphilic α-helical structure, which is exemplified by cecropin that is the antibacterial peptide identified in insects.
There is a hypothesis saying that the peptic ulcer is developed by stress and gastric hyperacidity. However, since it was found out that the peptic ulcer is caused by Helicobacter pylori (Blaser, M J., Trends Microbiol., 1, 255-260, 1991), interests have been focused on Helicobacter pylori. Helicobacter pylori is a Gram-negative bacterium which is an anaerobic microorganism that grows very slow and has a spiral body and a flagellum. Among many proteins that Helicobacter pylori produces, RPL1 is the protein that is composed of 230 amino acids and has a cecropin-like structure at the amino-terminal region. Particularly, 8 of those amino acids were identified as same as cecropin. The RPL1 amino-terminal region of Helicobacter pylori has the complete amphiphilic spiral structure (Putsep, K. et al., Nature, 398, 671-672, 1999). This amphiphilic peptide has the structure similar to that of the lipid component of cell membrane, so that its mechanism to destroy cell membrane of a microorganism by combining with lipid of the cell membrane or to destroy the microorganism itself by changing electric potential of cell membrane is possible. In addition, there is a report that the RPL1 amino-terminal region of Helicobacter pylori has the antibacterial activity (Putsep K. et al., Nature, 398, 671-672, 1999).
Accordingly, studies have been focused on the antibacterial activity of the amphiphilic peptide and thereby attempts have been made to develop an antibiotic by using the same. The amphiphilic peptides that have been identified so far are HP (2-20) peptide represented by SEQ. ID. NO: 1 and melittin (ME) peptide.
The said HP (2-20) peptide represented by SEQ. ID. NO: 1 that is the peptide confirmed to have the antibacterial activity along with the amphiphilic activity at the amino-terminal region of RPL1 protein originated from Helicobacter pylori has no cytotoxicity but has the antifungal activity together with the antibacterial activity (Biochem. Biophys. Res. Commun., 2002, 291, 1006-1013, Biochem. Biophys. Acta. 2002, 1598, 185-194).
The said melittin peptide is the peptide taking at least 50% of the solid components of bee venom, wherein the carboxy-terminal region is amidated. According to the previous reports, melittin has high cytotoxicity against eukaryotic cells so that it destroys animal cells so well even at a low concentration and at the same time displays the high antibacterial activity against such microorganisms as Gram-negative bacteria and Gram-positive bacteria (Habermann, E., Science, 177: 314, 1972; Steiner, H., et al., Nature, 292: 246, 1981; Tosteson, M. T., et al., Biochemistry, 228: 337, 1987).
The amphiphilic peptide belonging to cecropin family and having the amino acid sequence similar to that of HP (2-20) was first identified in drosophila and then later identified in silkworm pupa and pig small intestine as well. Particularly, cecropin A (CA) has the high antibacterial activity but the low antifungal activity and the low anticancer activity (Boman, H. G. and Hultmark, D., Annu. Rev. Microbiol., 41: 103, 1987).
In addition to the studies on the activity of the amphiphilic peptide, the studies on the amino acid sequence and the protein structure of the peptide have also been made. As a result, it was confirmed that the peptide has the sequence that is believed to be closely related to the antibacterial activity. Therefore, it is possible to prepare a novel synthetic peptide having the excellent antibacterial, antifungal, or anticancer activity by using the amino acid sequence of the amphiphilic peptide, precisely by substituting a specific sequence region with similar amino acids or by recombining a part of the sequence to produce a conjugation peptide, or by relocating a certain functional group of the peptide sequence (Chan, H. C., et al., FEBS Lett., 259: 103, 1989; Wade, D., et al., Int. J. Pept. Prot. Res., 40: 429, 1992).
Actually, the synthetic peptides mag A and mag G having anticancer effect were prepared by using the said amphiphilic peptides and their effects have been reported (Ohsaki, et al., Cancer Res., 52: 3534, 1992). In addition, the synthetic peptides showing antifungal activity were also prepared by combining the amino acids in the amphiphilic region, flexible region, and hydrophobic region of the magainin 2 and the melittin peptides, which were confirmed to work against bacteria and fungi strains (Korean Patent No. 0204501).
The present inventors previously prepared the antibiotic peptide (SEQ. ID. NO: 2) with improved antibiotic effect resulted from the increased hydrophobicity by substituting a specific amino acid of the conventional HP (2-20) with tryptophane, which was patent-registered (Korean Patent No. 0459808). In addition, the present inventors prepared another antibiotic peptide having the amino acid sequence represented by SEQ. ID. NO: 3 which has the folding structure made by replacing glutamate of the HPA3 peptide (SEQ. ID. NO: 2) with proline, which was also patent-registered (Korean Patent No. 0935029).
In general, the antibiotic peptide has a linear helical structure, which is able to attack cell membrane. At this time, if a folding is generated in a proper region of the linear helical structure, the ability to attack cell membrane increases. Therefore, it is possible to regulate the binding of the peptide onto the cell membrane by using the hydrophobicity or electric charge on the surface of the bacterial membrane or fungal membrane.
The present inventors tried to develop a novel natural antibacterial agent. As a result, the inventors synthesized the novel peptide represented by SEQ. ID. NO: 4 by replacing phenylalanine, the 12th amino acid of the peptide represented by SEQ. ID. NO: 3, with proline. Thereafter, the inventors confirmed that this novel peptide had a significant antibacterial activity but had no cytotoxicity, and also confirmed that this peptide of the invention had anti-inflammatory effect in vivo, so that the novel peptide could be used as an active ingredient of a natural antibacterial composition, leading to the completion of this invention.